125I标记外泌体在Pan02胰腺癌荷瘤小鼠体内的生物分布研究

目的 旨在开发用 ¹²⁵I-NaI标记外泌体的方法，并通过 γ 计数仪考察其在 Pan02 胰腺癌荷瘤小鼠体内的生物分布特征。方法 通过非放射性 NaI对用于治疗胰腺癌的工程化外泌体进行冷标记，采用透射电子显微镜、纳米粒子跟踪分析和Western blotting实验对标记前后外泌体进行表征。在此基础上采用Iodogen法进行外泌体的放射性 ¹²⁵I-NaI标记，分离纯化后测定 ¹²⁵I-NaI的标记率，Radio-HPLC法测定给药前后 ¹²⁵I-外泌体的放化纯度以考察其稳定性；将 ¹²⁵I-外泌体单次尾iv于Pan02胰腺癌荷瘤小鼠体内，分别于给药后2、6、24、72 h（每个时间点雌雄各3只）经CO₂麻醉后心脏放血处死小鼠，取血清、主要组织器官及肿瘤，γ计数仪测量其放射性计数，计算各组织/血清在不同时间点的蛋白沉淀率；并计算在不同时间点的每克组织（或每毫升血清）放射性计数占总注入放射性计数的百分比（%ID·g^-1或%ID·mL^-1）。结果 外泌体表征的结果显示，标记前后的外泌体形态一致，均成圆形或茶托样结构；标记前外泌体粒径峰值为 113 nm，标记后外泌体粒径峰值为 122 nm，粒径大小主要分布在 50～200 nm；均表达其标志性蛋白 CD63及 TSG101，符合外泌体特征。¹²⁵I-NaI标记外泌体的标记率为 27.82%，纯化后 HPLC法测得即时放化纯度为 100%，给药后放化纯度为（93.34±5.48）%。在小鼠尾 iv给药后 2 h，标记的外泌体主要分布在肝脏［（10.899 2±1.518 1）%ID·g^-1］和脾脏［（2.566 4±0.799 8）%ID·g^-1］，肿瘤中为［（0.291 0±0.056 0）%ID·g^-1］，脑、心脏、脂肪和肌肉组织摄取较少；给药后72 h，肝脏中仍有较高摄取，肿瘤中仍有放射性分布。给药后 2～6 h各组织脏器的蛋白沉淀率较低，表明 ¹²⁵I-NaI标记外泌体稳定性有所降低。结论 外泌体可以进行 ¹²⁵I标记，而且标记同位素前后对外泌体物理形态、生物学活性无明显影响；¹²⁵I标记外泌体的方法简便，标记率和放化纯度均较高；该外泌体产品在荷瘤小鼠体内大部分血流丰富的组织器官均有分布，且具有一定的肿瘤靶向定位能力。

Biodistribution of 125I labeled exosomes in Pan02 pancreatic cancer tumor bearing mice

Objective This study was aimed to develop a ¹²⁵I-NaI labeling method for exosomes, and to investigate their biological distribution in Pan02 pancreatic cancer tumor bearing mice by gamma counter.Methods Exosomes were cold-labeled with non-radioactive NaI. Exosomes were characterized before and after labeling by transmission electron microscopy, nanoparticle tracking analysis and Western blotting. On this basis, Iodogen method was used for radioactive ¹²⁵I-NaI labeling of exosomes, and the labeling rate of ¹²⁵I-NaI was determined after isolation and purification. Radio-HPLC method was used to determine the radiochemical purity of ¹²⁵I-exosomes before and after drug administration to understand its stability. ¹²⁵I-exosome was injected into Pan02 pancreatic cancer tumor bearing mice by a single caudal vein, and the mice were killed by cardiac bloodletting after CO₂ anesthesia at 2, 6, 24, 72 h (three males and three females at each time point). Serum, major tissues, organs and tumors were collected and their radioactivity counts were measured. The percentage (%ID·g^-1 or %ID·mL^-1) of the total injected radioactivity count per gram of each tissue and organ at different time points was calculated, so as to investigate the biological distribution of ¹²⁵I-NaI labeled exosomes in tumor-bearing mice.Results The morphology of exosomes before and after labeling was consistent with that of round or saucer-like exosomes. The peak particle size of exosomes was 113 nm before labeling and 122 nm after labeling, and the particle size was mainly distributed in the range of 50 ~ 200 nm. Both of them expressed their signature proteins CD63 and TSG101, which were consistent with exosome characteristics, indicating that isotope labeling did not affect their biological characteristics. The labeling rate of ¹²⁵I-NaI labeled exosomes was 27.82%, and the purity was 100% by HPLC after purification, and (93.34±5.48)% after administration. The labeled exosome was mainly distributed in liver [(10.899 2±1.518 1) %ID·g^-1] and spleen [2.566 4±0.799 8)% ID·g^-1] at 2 h after administration in the tail vein of mice. Meanwhile, (0.291 0±0.056 0) %ID·g^-1 was found in tumor. After 72 h of administration, there was still high uptake in liver and radioactive distribution in tumor. The protein precipitation emissivity of various tissues and organs was low 2 ~ 6 h after administration, indicating a decrease in the stability of ¹²⁵I-NaI labeled exosomes.Conclusion Extracellular vesicles can be labeled with ¹²⁵I, and there is no significant effect on their physical morphology and biological activity before and after isotope labeling. The method of labeling exosomes with ¹²⁵I is simple, with high labeling rate and radiochemical purity. This exosomes product is distributed in most blood rich tissues and organs of tumor bearing mice, and has certain tumor targeting and localization capabilities.